New biologic (Ab-IPL-IL-17) for IL-17-mediated diseases: identification of the bioactive sequence (nIL-17) for IL-17A/F function

Objectives Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therapeutic efficacy and immunogenicity for subgroups of patients or IMIDs reduce their clinical use. Therefore, there is an urgent need to improve the coverage and efficacy of antibodies targeting IL-17A and/or IL-17F and IL-17A/F heterodimer. Methods and results Here, we initially identified a bioactive 20 amino acid IL-17A/F-derived peptide (nIL-17) that mimics the pro-inflammatory actions of the full-length proteins. Subsequently, we generated a novel anti-IL-17 neutralising monoclonal antibody (Ab-IPL-IL-17) capable of effectively reversing the pro-inflammatory, pro-migratory actions of both nIL-17 and IL-17A/F. Importantly, we demonstrated that Ab-IPL-IL-17 has less off-target effects than the current gold-standard biologic, secukinumab. Finally, we compared the therapeutic efficacy of Ab-IPL-IL-17 with reference anti-IL-17 antibodies in preclinical murine models and samples from patients with RA and IBD. We found that Ab-IPL-IL-17 could effectively reduce clinical signs of arthritis and neutralise elevated IL-17 levels in IBD patient serum. Conclusions Collectively, our preclinical and in vitro clinical evidence indicates high efficacy and therapeutic potency of Ab-IPL-IL-17, supporting the rationale for large-scale clinical evaluation of Ab-IPL-IL-17 in patients with IMIDs.

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Isolation of human peripheral blood lymphocytes (PBLs) and neutrophils
Venous blood from healthy volunteers was collected into 10 ml EDTA coated tubes.Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation of blood on histopaque 1077 (8), and PBL were prepared by panning human PBMC on culture plastic to remove monocytes (9,10).Isolated cells were washed, counted, and adjusted to a final concentration of 1×10 6 /ml in Medium 199, supplemented with 0.15% BSA (Sigma-Aldrich; M199BSA).On a few occasions, neutrophils were isolated using a two-step density gradient with histopaque 1077 and histopaque 1119 as previously described (11)(12)(13) and suspended at 1×10 6 /ml in M199BSA, after hypotonic lysis of contaminating erythrocytes.

Isolation of human fibroblasts
Synovial tissue samples were obtained by ultrasound-guided biopsy (14) from treatment naïve patients with a new onset of clinically apparent arthritis and a symptom duration of ≤12 weeks who at follow-up had resolving arthritis (Res) (15).Patients were classified as having resolving arthritis if there was no clinical evidence of synovial swelling at any peripheral joint (out of a swollen joint count of 66 joints) on final examination at least one year after initial presentation, in the absence of disease modifying anti-rheumatic drugs or glucocorticoid therapy for at least the previous 3 months (16).Alternatively, synovial tissue samples were collected from subjects with established, treated RA undergoing joint replacement surgery (RA).RA was classified according to 1987 American College of Rheumatology criteria (17) (Table 2).Fibroblasts, used between passages 4-6 (18), were isolated as previously described (19) and stimulated for 24 h at 37°C with IL-17 (10 ng/ml) in combination with TNF-α (100 U/ml).In IL-17 neutralising antibodies experiments, cells were also pre-treated with neutralising antibodies MAB317 or Ab-IPL-IL-17™ (10 µg/ml) 30 min before stimulation with IL-17 and TNF-α (20).Samples were analysed by Elisa (see below).

Ex vivo whole blood assay
Whole blood culture and stimulation were performed as previously described with small modifications (29).Briefly, venous blood collected into lithium heparin tubes from patients with clinically diagnosed IBD (Table 3) was placed into 96-well plates with or without Ab-IPL-IL-17™ (10 µg/ml) at 37 °C, 5% CO2 for 4 h.Following incubation, samples were centrifuged (300 g, 5 min at RT), and supernatants were collected and stored at −80 °C until IL-17A levels measurements by Elisa assay (30).
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Elisa and Elisa Spot assays
The levels of IL-6 (DY406 and DY206, respectively, mouse and human kits) and TNF-α (DY210) in the in vitro and ex vivo supernatants were measured at 2 h or 24 h using commercially available enzyme-linked immunosorbent assay kits (Elisa kit, R&D System) according to the procedure previously described (25).Briefly, 100 μl of supernatants, diluted standards, quality controls, and dilution buffer (blank) were applied on a plate with the monoclonal antibody for 2 h.After washing, 100 μl of biotin-labelled antibody was added, and incubation continued for 1 h.The plate was washed, and 100 μl of the streptavidin-HRP conjugate was added, and the plate was incubated for a further 30 min period in the dark.The addition of 100 μl of the substrate and stop solution represented the last steps before the reading of absorbance (measured at 450 nm) on a microplate reader.Antigen levels in the samples were determined using a standard curve and expressed as pg/ml (31,32).Similarly, serum level of IL-17A, IL-17F, total IgG and IgG1 (Elisa kit, R&D System) were measured at indicated time-points for in vivo neutralisation and immunogenicity assays.
Inflammatory exudates from the air pouch were incubated with the precoated proteome profiler array membranes according to the manufacturer's instructions (ARY006, R&D System).Dot plots were detected using the enhanced chemiluminescence detection kit and Image Quant 400 GE Healthcare software (GE Healthcare, Italy) and successively quantified using GS 800 imaging densitometer software (Bio-Rad) as extensively described (33,34).

Haematological investigations
Standard laboratory procedures were used for blood sampling and measurements (34).Haematological investigations, for all experimental conditions, including blood count test, leukocyte, and sidereal formula were performed on citrated and not-anticoagulated blood samples, respectively.Serological tests were performed by CELL-DYN Sapphire purchased from Abbott S.R.L. (Milan, Italy).All procedures were conducted under strictly aseptic conditions.

Flow Cytometry
HDBEC and hMDMs, collected after 24 h of treatment, were washed with PBS without Ca 2+ and Mg 2+ containing 25 mM lactose for 20 min at RT with occasional mixing.Cells were then incubated with FcR blocking agents (Miltenyi Biotec) in PBS without Ca 2+ and Mg 2+ containing lactose before staining cells with antibodies IL-17 RA (1:100) and IL-17 Receptor C (1:100).Moreover, HDBEC cells were also stained with ICAM-1 (1:50; clone HA58; Thermofisher scientific) and VCAM-1 (1:50; BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) clone 51-10C9; Thermofisher scientific).Protein expression was analysed by flow cytometry on BD LSRFortessa™ x-20 (BD, Biosciences, London, UK), and data were analysed using MRFlow and FlowJo software operation.The unspecific binding of antibodies was quantified by using corresponding isotype controls, gating strategy in Fig. S12 (3,35).

Sample processing for flow cytometry analysis
Mice were culled by cervical dislocation and lower limbs dissected and enzymatically digested as previously described (24).Briefly isolated joints were incubated in an enzyme cocktail consisting of 100g/ml Collagenase D (Roche) and 100g/ml DNase I (Sigma) diluted in RPMI containing 2% FBS at 37°C for 45 min.Undigested tissue was incubated with this enzyme cocktail again for an additional 30 min before cell suspension was passed through 100M cell strainer (Vicedeal).Samples were washed in RPMI containing 2% FBS at 400g for 5 min.Cells were incubated in the RBC lysis buffer (Sigma) for 4 min at room temperature, washed at 400g for 5 min in PBS and resuspended in MACS buffer.All samples were blocked with FcR blocker (Biolegend) for 15 min prior to staining with the following of antibodies and Zombie Aqua (BV510) (all from Biolegend) for 20 minutes at 4°C prior to washing and fixation with 2% PFA: Anti-Cd11b BV650 (clone M1/70), anti-CD45 BV605 (clone: 109841), anti-Ly6C FITC (clone: 128005), anti-F4/80 APC-cy7 and anti-Ly6G APC (clone:IA8).Compensation controls were generated by killing cells under hot water for 1 minutes and adding zombie aqua to it for 15 minutes.After 15 minutes live spleen cells were added and run on fortessa.Samples were acquired using Fortessa-X20 and data analysed offline using FlowJo (V-10.2.6).

Elisa-based binding assay
Murine or Human IL-17RA-Fc (4481-MR or 177-IR, R&D System), and IL-17RC-Fc (2270-ML or 9284-MR, R&D System) were plated in 96 well plates at concentration of 2 µg/ml.Plates were incubated overnight at 4 ˚C, and then washed 3 times with TBST wash buffer and blocked for 1 h with 200 µl of protein-free T20 blocking buffer.Cytokines were biotinylated with EZ Link NHS-Biotin according to the manufacturer's instructions.Biotinylated cytokines and proteins (0-750 ng/ml) were incubated alone or in the presence of IL-17 neutralising antibodies (MAB421/MAB317 or Ab-IPL-IL-17™; 0-750 ng/ml) in wash buffer for 1 h.Plates were incubated for 30 min at RT before 3 washes with TBST and a 30 min incubation with Streptavidin-HRP.The colorimetric signal was developed with TMB, and the reaction stopped with sulfuric acid (stop solution).OD values were read on a SpectraMax M3 (Molecular Devices).The percentage of binding or inhibition was calculated as previously described (36).

Human PBMC transendothelial migration assay
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) HDBEC seeded into 12-well plates were stimulated with TNF-α or IL-17 alone or in combination with IL-17 neutralising antibodies for 24 h, as previously described (35).Monolayers were washed with M199 containing 0.15% w/v BSA.PBMC (1 × 10 6 ) were allowed to adhere and migrate on stimulated HDBEC monolayers at 37 °C for 20 min.Non-adherent cells were removed by washing in M199 BSA, prior to the adherent cells being fixed with 2% glutaraldehyde (Sigma-Aldrich) for 15 min.Five random fields of view were imaged per well using phase-contrast microscopy (IX71; Olympus, Tokyo, Japan).Images were analysed offline using Image Pro 7 software (Media Cybernetics, Rockville, MD, USA), with adherent cells being defined as phase bright round cells, whilst transmigrated cells were shape changed and phase dark.PBMC adhesion was expressed as total number cells per mm 2 .Transmigration was expressed as a percentage of adherent cells (11).

Static adhesion assay on PBL
HDBEC were seeded and stimulated with TNF-α (100 U ml -1 , R&D System) and/or IFN-γ (10 ng ml -1 , Peprotech) in presence of MIE extract (0.1-10 μg ml-1) or its corresponding vehicle (DMSO, 0.25%) for 4 h or 24 h before the assay with neutrophils or lymphocytes, respectively.PBL and neutrophils were isolated from healthy volunteers as described above prior to being added to activated HDBEC for 7 min at 37°C.Wells were washed twice with PBS to remove unbound cells and fixed with 2% glutaraldehyde for 10 min at RT. Glutaraldehyde was removed with several 1 ml washes of PBS (with Ca 2+ and Mg 2+ ).Images were taken at five different fields in the centre of each well using phase-contrast microscopy (IX71; Olympus, Tokyo, Japan).Images were analysed offline using Image-pro 7.0 software (Media Cybernetics, Rockville, MD, USA), with adherent cells being defined as phase bright round cells, whilst transmigrated cells were shape changed and phase dark.PBL and neutrophils adhesion was expressed as the total number of cells per mm 2 , while transmigration was expressed as a percentage of adherent cells (35).

Peptide synthesis and purification
Peptides were synthesized manually by the ultrasonic-assisted solid-phase peptide synthesis (US-SPPS), via the Fmoc/tBu orthogonal protection strategy (37).In brief, Fmoc-deprotection (20% piperidine in DMF, 0.5 + 1 min treatments) and coupling (HBTU/HOBt as activating/additive agents, 5 min treatment) reactions were cyclically performed by ultrasonic irradiation to generate the resinbound target peptide sequence.As solid supports, the 2-chlorotrityl chloride (2-CTC) (0.1 mmol from 1.70 mmol/g as loading substitution) or the Rink amide (0.1 mmol from 0.72 mmol/g as loading substitution) resins were employed depending on the C-terminal target as carboxylic acid or amide, respectively.Upon cleavage, crude peptides were purified by reverse-phase HPLC (RP-HPLC) using linear gradients of MeCN (0.1% TFA) in water (0.1% TFA), from 10 to 90% over 30 min, with a flow rate of 10 mL/min and UV detection at 220 nm.Final products were obtained by lyophilisation of the appropriate fractions after removal of the MeCN by rotary evaporation.All compounds examined for biological activity were of >97% purity, and the correct molecular ions were confirmed by mass spectrometry.

Circular dichroism
CD experiments were performed on a Jasco J-815 spectropolarimeter equipped with a PTC-423S/15 Peltier temperature controller.Cells with 0.1 cm path length and peptide concentration of 0.1 mM were used to record CD spectra between 200 and 260 nm, with a 1 nm bandwidth and a scan rate of 20 nm/min.Spectra were recorded at 20 °C.Spectra were signal-averaged over three scans, baseline-corrected by subtracting a buffer spectrum, and smoothed using the meansmovement function.Spectra were analyzed for secondary structure composition using the BeStSel method (38).
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)  (39)(40)(41) was used to predict the nIL-17™ structure from the amino acid sequence of the peptide.The primary sequence is submitted using the single letter code and the program predicts the 3D conformation by assembling predicted conformations of short local sequences using a greedy procedure driven by a coarse-grained energy score.PEP-FOLD 4 uses a new version of the force field (sOPEP2) that makes use of a Mie representation instead of the former Van der Waals representation for non-bonded interactions, and also includes an energy term using the Debye-Hueckel formalism to model pH, ionic strength dependence, and extremity blocking.

Molecular Docking
All docking calculations were conducted using the Schrödinger molecular modeling suite (version 2021-4).Protein structures were obtained from the Protein Data Bank (PDB).In particular, the structure of the complex of IL-17A (monomers A and B) with IL-17RA and IL-17RC receptors (PDB id: 7ZAN) (42) was selected to perform molecular docking studies with nIL-17™ peptide.The IL-17RA and IL-17RC receptors and nIL-17™ were prepared with the aid of the Protein Preparation Wizard panel of Maestro Suite (43) adding the missing hydrogen atoms and removing any water molecule with less than two hydrogen bonds to non-water molecules.In addition, the side chain ionization and tautomeric states were predicted, and the H-bonding network of the receptor refined minimizing the position of each hydrogen.Receptor grid generation tool of Glide software was used to generate the search grid around the IL-17A C-terminal region of monomers A and B to perform docking simulations of IL-17RA and IL-17RC, respectively (44)(45)(46).Then, docking calculations were performed using Glide 9.4 in its SP-peptide variant and employing the OPLS4 force field (44)(45)(46).Thus, the top-ranked complexes were selected and visually checked for a good chemical geometry.

Immunization and fusion protocols
The immunization protocol and the synthesis of a novel IL-17 neutralising antibody (Ab-IPL-IL-17™) were commissioned to ProteoGenix (France).All experimental details and procedures are reported in the Patent application (IT patent No. 102022000016722).
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Fig. S5 -Auxiliary Table 2 .
Fig. S5-IV.(A)Docking-predicted binding mode of nIL-17™ (light blue) to IL-17RC receptor (violet) (PDB id: 7ZAN), and (B) zoom view of the binding mode highlighting the H-bond interactions.Specifically, H18, H19, and A21 of C-terminal region of nIL-17™ form H-bond interactions with W207, R284, T285, and N286 of IL-17RC D2 domain.In addition, V13 of nIL-17™ forms a H-bond to D281 side chain of the same receptor domain, as in the case of IL-17RA.The interactions established by N-terminal region of nIL-17™, L1 and E2 can establish two H-bond interactions with D193 and R195 of the linker between D1 and D2 domains, respectively.
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